In Vitro Antimicrobial Potential of the Lichen Parmotrema sp. Extracts against Various Pathogens.

OBJECTIVE(S)
The ongoing increasing antibiotic resistance is one of the biggest challenges faced by global public health. The perennial need for new antimicrobials against a background of increasing antibiotic resistance in pathogenic and opportunistic microorganisms obliges the scientific community to constantly develop new drugs and antimicrobial agents. Lichens are known prolific sources of natural antimicrobial drugs and biologically active natural products. This study was aimed to explore in vitro antimicrobial activity of lichen Parmotrema sp.


MATERIAL AND METHODS
The methanol and aqueous extracts of lichen Parmotrema sp. was extracted using Soxhlet extractor. Antibiotic assessment of methanol and aqueous extracts was done against eight bacterial (Escherichia coli, Staphylococcus aureus, Proteus mirabilis, Salmonella sp., Shigella sp., Enterococci faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae,) clinical pathogens and five plant pathogenic fungal strains (Aspergillus terreus strain JAS1, Scedosporium sp. JAS1, Ganoderma sp. JAS4, Candida tropicalis and Fusarium sp.) by Kirby-Bauer method.


RESULTS
The methanol lichen Parmotrema sp. extract inhibited all the test organisms. The highest antibacterial activity was found against Pseudomonas aeruginosa and Staphylococcus aureus. The weakest activity was manifested in Salmonella sp. and Scedosporium sp. JAS1. Strong antifungal effect was found against Ganoderma sp. JAS4 and Fusarium sp. The aqueous lichen Parmotrema sp. extract revealed neither antibacterial nor antifungal activity.


CONCLUSION
The present study shows that tested lichen Parmotrema sp. extracts demonstrated a strong antimicrobial effect. That suggests the active components from methanol extracts of the investigated lichen Parmotrema sp. can be used as natural antimicrobial agent against pathogens.


Introduction
Lichens are symbiotic organisms composed of a fungal partner (mycobiont) in association with one or more photosynthetic partners (photobiont). The photobiont can be green algae, cyanobacteria, or both (1). They usually grow on rocks, non-fertile ground, as well as epiphytes on the trees and leaves (2). Lichens have also, for hundreds of years, been used in many countries as a cure for diseases of humans (3). In many European countries numerous species have been used for treatment of stomach diseases, diabetes, whooping cough, pulmonary tuberculosis, cancer treatment and skin diseases (4). The usage of lichens for many years in the traditional medicine was later justified by numerous researches that confirmed their various biological activities.
Lichens produce secondary metabolites called the "lichen substances," which comprise depsides, depsidones, dibenzofurans, xanthones and terpene derivatives. These metabolites sometimes make more than 30% of the dry mass of thalus (3). Various biological activities of lichens and their metabolites are known, such as antiviral, antibiotic, antitumoral, antiallergic, antiherbivoral and they inhibit growth of plants as well as various enzymes (5)(6). It has been observed that "Lichens extracts" and "Lichen substances" produce antimicrobial agents (3)(4)(7)(8)(9) and continuous and uncontrolled use of synthetic drugs has led to the need to find new preparations of natural product drugs (3).
Bioactive natural products have more beneficial effects on organism when compared to synthetic drugs. By considering the multi-drug resistant to pathogens towards infectious diseases, this study was carried out to screen for antimicrobial agents from natural origin.

Antibiogram
The multi-drug resistant bacterial test pathogens were screened against standard antibiotic disc, vancomycin (30 mcg/disc), tigecycline (15 mcg/disc), erythromycin (15 mcg/disc), ciprofloxacin (30 mcg/disc), penicillin (10 mcg/disc), ofloxacin (5 mcg/disc) and fungal isolates were screened against flucanazole (25 mcg/disc) and voriconazole (5 mcg/disc). Antibiotic sensitivity test was performed by Kirby-Bauer method on Muller-Hinton agar plates (10). Bacterial test pathogens were lawn cultured on Muller-Hinton agar plates using sterile cotton swabs. The standard antibiotic discs were placed on it using sterile forceps. Plates were incubated at 37°C for 24-48 hr and were observed for zone of inhibition. Fungal test pathogens were seeded into Potato Dextrose Agar petridishes, antibiotics disc was placed on it using sterile forceps. Plates were incubated at 30°C for 5 days and were observed for zone of inhibition.

Antimicrobial assay
The sensitivity of microorganisms to methanol and aqueous extracts were tested by measuring the zone of inhibition of given concentration of lichen Parmotrema sp. extracts by the well-diffusion method. Clinical bacterial isolates were swabbed onto Muller-Hinton agar plates. Four wells were punctured onto the agar plate. 50 mg/ml of dry lichens extract with different concentrations (25 µl, 50 µl, 75 µl and 100 µl) were loaded into the wells. The petriplates were incubated for 24 hr, and the zone of inhibition was measured around the wells.
For antifungal activity, the appropriate fungal test pathogens were seeded in potato dextrose agar (PDA) in petridishes (11). Paper disks of 6 mm in diameter were laid on the inoculated test organism after being soaked with 15 µl of the lichen extract of 50 mg/ml, antimicrobial activity was determined by measuring the zone of inhibition around the disk. Streptomycin was used as positive control of the growth in bacteria and flucanazole in case of fungi. DMSO (dimethyl sulfoxide) solution was used

Antimicrobial assay
The antimicrobial activity of methanol and aqueous extracts of lichen Parmotrema sp. against the test microorganisms was estimated based on presence or absence of inhibitory zones and their diameter value of the extract. Antibacterial activity of lichen extracts is shown in Table 1 and antifungal activity in Table 2. this study showed strong antimicrobial action on the test pathogens, which usually depends on the species of lichen, the type of extracting solvent used and the concentration of lichen extract. Similar differences were observed by other authors (9,(12)(13)(14). Aqueous extracts manifested no activity in relation to the microorganisms tested. Land